JP2884045B2 - Iron-lactoferrin complex and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to carbonic acid and / or bicarbonate which are stable over a wide pH range and exhibit heat resistance.
The present invention relates to an iron-lactoferrin complex and a method for producing the same. The carbonate and / or bicarbonate-iron-lactoferrin complex of the present invention eliminates the unique astringent taste of iron and prevents the promoting action of peroxide production of iron, thereby preventing or treating anemia.
It is useful as a raw material for foods, pharmaceuticals, feeds, cosmetics, etc. for the purpose of iron enhancement or prevention of pathogen adherence.

[0002]

2. Description of the Related Art Lactoferrin (hereinafter referred to as Lf) is known to have various physiological functions such as promotion of iron absorption, suppression of lipid peroxide production, antibacterial activity, antiviral activity, cell growth, and control of the immune system. Therefore, foods, pharmaceuticals, feeds containing Lf,
Various attempts have been made to manufacture cosmetics and the like. Many of these products may be heat sterilized or may come in contact with boiling water during use. However, Lf is thermally unstable, and has a fatal disadvantage that it precipitates upon heat denaturation, loses its iron binding ability, and loses its physiological function.
Therefore, a study on the thermal stabilization of Lf was performed, and it was reported that Lf heated at pH 4 had the same ability to bind iron as raw Lf (Davidson and Lonnerdal, Am.
J. Physiol. 257: G930-G934, 1989). However, the Lf thus prepared has a disadvantage that it gradually denatures during storage and loses its physiological function. It is also known that heating Lf under conditions of low ionic strength can maintain physiological activity (Japanese Patent Laid-Open No. 4-108629). But,
The ionic strength is not necessarily low in the actual product system. Therefore, the effect of the relationship between pH and electric conductivity Ω on the thermal stability of Lf was examined, and log Ω ≦ (2.96 / pH) +0.64 (pH <5) log Ω ≦ (29.37 / pH) −4.62 (5 ≦ pH ≦ 7.9) log Ω ≦ −0.917 (pH> 7.9)
A method has been developed for stabilizing the thermal stability of the polymer (Japanese Patent Laid-Open No. 4-8269). However, when this condition is not satisfied, the Lf solution adjusted to this condition and another raw material solution are separately sterilized,
It is necessary to take aseptic means of mixing the two.
However, according to the "Ministry of Health and Welfare Ordinance on Milk and Dairy Products", it is specified that all ingredients are mixed and then heat-sterilized, and sterilization separately is not practical. Therefore, the present inventors have further studied and found that it is possible to impart heat resistance by binding and adsorbing a sufficient amount of iron to Lf, and filed a patent application for the production method thereof ( Heat-resistant lactoferrin-iron conjugate and method for producing the same (JP-A-6-239900). However, this invention also has the following disadvantages. 1. The produced Lf precipitates under the environment of pH 7.1 or more. 2. The produced Lf is soluble and heat-resistant in an environment of pH 6.5 or more and pH 7 or less, but if the iron bound to Lf exceeds 150 molecules per Lf molecule, the pH after production is increased. When stored in an environment of pH 6.5 or more and pH 7 or less, it gradually becomes insoluble.

[0003]

SUMMARY OF THE INVENTION The present invention has been made to improve such a disadvantage.
That is, an object of the present invention is to provide an iron-Lf composite which is stable for a long time in a wide pH range and has heat resistance, and a method for producing the same.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above problems. 1) Lf was added to a solution containing carbonate ions or bicarbonate ions.
A carbonate or bicarbonate-iron-Lf complex is formed upon addition and mixing of solutions containing irons and iron ions; 2)
This complex is formed for the first time by taking a measure of adding a solution containing a specific ratio of Lf and iron to carbonate or bicarbonate to a solution containing carbonate or bicarbonate, The resulting complex containing a high proportion of iron is stable for a long time in a wide pH range, has heat resistance, eliminates the unique astringent taste of iron, and prevents the promoting action of peroxide generation of iron. To complete the present invention. That is, the present invention relates to a carbonate and / or bicarbonate ion-iron-Lf complex (hereinafter, referred to as iron) containing 15 to 1000 molecules of iron and 15 or more molecules of carbonate and / or bicarbonate per molecule of Lf. -Lf complex). Such an iron-Lf complex does not precipitate at room temperature for at least one month at a pH of 2.1 or more and 9.0 or less, does not precipitate even when heated, and has the property of having no iron-specific astringent taste.

[0005] Such a complex is composed of i) carbonic acid, or ii) bicarbonate, or iii) a solution containing carbonic acid and bicarbonate (A solution), and iv) a solution containing iron and v) lactoferrins ( B solution). At this time, the vi) iron ion molarity of the B solution is determined by vii) the solution (reaction solution) in which a part or all of the A solution and the B solution are mixed (reaction solution). ) Up to 1/3, preferably 1/10, more preferably 1/30, even 1/60, most preferably 1/100 of the molar concentration of carbonate and bicarbonate ions, and ix) The molar concentration of lactoferrins is
It must be from 1/15 to 1/1000 of the x) iron ion molarity of the B solution. The iron and the lactoferrin in the solution B may be prepared by mixing the solutions separately dissolved in the solution A, or may be separately mixed in the solution A. However, the iron solution must not be added before the lactoferrin solution. Further, the reaction needs to be performed in a solution, but either iron or lactoferrin to be added may be in a solid state. Furthermore, in order to keep the molar concentration of carbonate ions and bicarbonate ions in the reaction solution high, an insoluble amount of carbonic acid and / or bicarbonate may be contained in the A solution in advance, I) Carbonic acid and / or bicarbonate; and / or ii) Carbonic acid and / or bicarbonate during the addition of the B solution, ie, the reaction solution in the step where a part of the B solution is added to the A solution. A solution may be added.

When adding the B solution to the A solution,
The pH of the carbonic acid and / or bicarbonate solution of solution A and / or the carbonic acid and / or bicarbonate solution gradually decreases.
Occasionally, precipitation may occur during production. The pH of solution B is between 8 and 9, and the isoelectric point of Lf is also between pH 8 and 9 (EN Baker, Advanced In Organic Chemistry 41,390)
Thus, this phenomenon may indicate that Lf and / or the iron-Lf complex has undergone isoelectric precipitation. In order to avoid this, the pH of the mixed solution of the A and B solutions is maintained at a value higher than the isoelectric point of Lf, or the pH of the mixed solution of the A and B solutions is Lf and / or that of the iron-Lf complex. PH higher than the electric point
It is preferable to terminate the production, adjust the pH of the B solution to the isoelectric point of Lf or lower, or adjust the pH of the mixed solution to the isoelectric point or lower as needed during the addition of the B solution. By doing so, precipitation which is considered to be an isoelectric precipitation of Lf and / or iron-Lf complex is suppressed, and particularly in the case of production performed on an industrial scale, the iron-Lf complex can be produced more reliably and stably. can do.

[0007] The iron-Lf complex of the present invention comprises i) carbonic acid, or ii) bicarbonate, or iii) carbonic acid and bicarbonate.
And iv) a solution containing lactoferrins (A solution);
v) It is also obtained by mixing a solution containing iron (B solution). At this time, the vi) iron ion molarity of the B solution is determined by vii) a solution obtained by mixing the A solution and a part or all of the B solution ( Viii) 1/3 or less, preferably 1/1, of the molar concentration of carbonate ion and bicarbonate ion dissolved in the reaction solution).
0, more preferably 1/30, even more preferably 1/60, most preferably 1/100 or less, wherein the concentration of ix) lactoferrins in the A solution is 1/1 / x the molar concentration of x) iron ion in the B solution. Fifteen
Needs to be 1/1000. Also in this production method, it is preferable to use the above-mentioned method in order to prevent precipitation which rarely occurs near the isoelectric point.

The Lf compounds used in the present invention include lactoferrin isolated from secretions of milk of mammals such as humans and cattle, transferrin isolated from blood and organs, and ovotransferrin isolated from eggs and the like. There is. There are already a number of known methods for separating these in a large amount, but they may be separated by any method. Moreover, those produced from microorganisms, animal cells or transgenic animals by genetic manipulation may be used. Further, these Lf compounds may be those obtained by enzymatic decomposition. Lf does not need to be completely separated and may contain other components.

The amount of iron to be added is at least 15 moles, more preferably at least 30 moles, even more preferably at least 60 moles as iron ions per mole of Lf, and the upper limit is at most 1,000 moles, preferably at most 480 moles. . In order to shorten the time required for the production or not to decrease the yield, the amount is preferably 240 mol or less. If the amount of iron added exceeds the above upper limit, iron will precipitate. Examples of the iron agent to be used include iron salts having a pH of 4 or less when dissolved in deionized water, for example, mainly trivalent iron agents such as ferric chloride, ferric nitrate, and ferric sulfate. An iron salt having a pH of more than 4 when dissolved in deionized water, such as ferrous sulfate, cannot form an iron-Lf complex. At this time, even if the pH is lowered to 4 or less,
Lf complexes cannot be formed.

Next, the difference in thermal stability depending on the type of the iron agent will be described in a test example.

[Test Example 1] (Material) (Solution A) containing 1 mol / L sodium bicarbonate
1 liter of pH 8.3 solution (B1 solution) A solution containing 5 mmol of various iron agents as iron
0.2 liter (B2 solution) Lf (manufactured by Oleofina Co., Ltd.) A solution containing 33 micromoles 0.8 liter After mixing the B1 solution and the B2 solution (B solution), the B solution was added to the A solution to prepare an iron-bound Lf. After desalting and concentrating this solution with an ultrafiltration membrane with a molecular weight of 5,000, the final concentration
Dilute to a concentration of 3.6 mmol / L iron with a buffer simulating liquid food of pH 7.5 containing 0.05 mol / L imidazole and 0.15 mol / L salt (simulated buffer), and heat at 90 ° C for 10 minutes. , Lf precipitates were observed. Table 1 shows the results.

[0011]

[Table 1] Iron agent 120μg / ml of iron is deionized Precipitation in water pH when dissolved pH Fe (NO ₃ ) ₃ 2.7 without FeSO ₄ 4.8 with FeCl ₃ 2.7 without FeCl ₂ 4.6 with Fe (SO ₄ ) NH ₄ 4.8 with Fe ₂ (SO ₄ ) ₃ 2.6 without iron pyrophosphate 6.8 with iron citrate 6.5 Yes Iron fumarate 4.5 Yes FeSO ₄ 4.8 → 2.7 (pH lowered with hydrochloric acid) Yes ─────────────────────────── ───────── Thus, the iron-Lf complex of the present invention cannot be formed unless an iron agent having a pH of 4 or less when dissolved in deionized water is used. Further, when the pH when dissolved in deionized water exceeds 4, even if the pH is adjusted to 4 or less, the iron-Lf complex of the present invention cannot be formed.

As the A solution containing carbonic acid or bicarbonate,
Examples thereof include carbonated water, ammonium bicarbonate, sodium bicarbonate, potassium bicarbonate, sodium carbonate, calcium carbonate solution, and a mixed solution thereof. Sodium hydroxide, ammonia, potassium hydroxide, hydrochloric acid, citric acid, lactic acid and the like can be mixed and used as pH adjusters. The A solution may contain other substances, for example, sugar, protein, fat, and the like.

It is important, however, that the ratio of the concentration of iron ions during the reaction to the concentration of carbonate ions and bicarbonate ions is lower than that of iron ions.
This is a point that affects the thermal stability of the Lf composite. Test examples relating to this will be described.

Test Example 2 (Materials) (A solution) 1 liter of solution containing sodium bicarbonate of each concentration (B1 solution) 0.2 liter of solution containing ferric chloride (B2 solution) 0.8 liter of solution containing 1 mmol of Lf 0.8 ml of B1 solution The B2 solution was mixed to prepare a solution B, and 1 liter of the B solution was added to the A solution to prepare an iron-bound Lf. The solution B was also diluted with deionized water. To make the final molar concentration of bicarbonate ion 0.6 or more, sodium bicarbonate was added at the time of mixing the solution A and the solution B, or a necessary amount of sodium bicarbonate was added to the solution A in advance to obtain a saturated solution. This solution was hydrodesalted with an ultrafiltration membrane having a molecular weight of 5,000 and further concentrated. Then, the mixture was diluted with a simulated buffer solution to an iron concentration of 3.6 mmol / liter, heated at 90 ° C. for 10 minutes, and observed to form Lf precipitate. Table 2 shows the results. Note that the minimum bicarbonate iron ion ratio in the table was calculated by dividing the bicarbonate ion molar concentration after mixing the A solution and the B solution by the iron ion molar concentration of the B solution.

[0014]

Table 2 When B1 solution contains 30 mmol of ironモ ル Molar concentration of bicarbonate ion Minimum 有無 Presence of precipitation Bicarbonate ion / iron ion ratio A solution After AB mixing (molarity / molarity) ────────────────────────────── 1.0 0.5 None 16.67 0.8 0.4 None 13.33 0.6 0.3 None 10.00 0.4 0.2 Slight 6.67 0.2 0.1 Slightly 3.33 0.1 0.05 Yes 1.67 ────────────────────────────────────

[0015]

Table 3 When the B1 solution contains 200 mmol of iron and the B solution is diluted 20 times (the B solution contains 10 mmol / L of iron).モ ル Molar bicarbonate ion minimum 最小 Precipitation Presence Bicarbonate ion / iron ion ratio A solution After AB mixing (molarity / molarity) ────────────────────────────── ───── Saturation Saturation None 1 2 0 or more Saturation 1.2 None 1 2 0 Saturation 1.0 None 1 0 0 Saturation 0.8 Very little 8 0 Slightly present 6 0 1.0 0.5 Slightly present 5 0 0.6 0.3 Slightly present 3 0 0.3 0.15 Yes 1 5 ──────────────────────────── ───────

As described above, as the iron concentration of the B solution is increased, it is necessary to increase the bicarbonate ion concentration of the AB mixed solution. As described above, in the preparation of the iron-Lf complex of the present invention, in the process of adding the solution B to the solution A, at least 3 molecules, preferably 10 molecules or more, of carbon around one molecule of the added iron. And / or the bicarbonate molecule must always be present. In addition, when more than 200 iron molecules are bonded to one Lf molecule, 30 or more, preferably 60 or more, more preferably 100 or more, carbonate ions and / or 100 or more molecules of iron are surrounded around one iron molecule. Alternatively, bicarbonate ion molecules may be present.

Next, the following study was conducted on the amount of carbonic acid and / or bicarbonate bound to the iron-Lf complex of the present invention.

Test Example 3 (Material) (A solution) 1 liter of solution containing sodium bicarbonate of each concentration (B1 solution) 0.2 liter of solution containing 100 mmol of ferric chloride (B2 solution) 0.8 liter of solution containing 1 mmol of Lf B1 After mixing the solution and the B2 solution (B solution), the B solution was added to the A solution, and the mixture was slowly stirred at 4 ° C. and 37 ° C. for each reaction time to prepare Lf to which iron was bound. This solution was diluted with a simulated buffer to an iron concentration of 3.6 mmol / L, heated at 90 ° C. for 10 minutes, and observed to form Lf precipitate.
Furthermore, the solution after the reaction for 192 hours was separated into iron-bound Lf and the aqueous solution using an ultrafiltration membrane with a molecular weight cut-off of 5000, and the carbonate and bicarbonate ion concentrations in the solution permeating through the membrane were analyzed by ion chromatography. did. The results are shown in Tables 4 and 5.

[0018]

[Table 4] When reaction temperature is 4 ℃ ℃ Minimum bicarbonate filtration Membrane permeation Bicarbonate ion / Carbon bicarbon in liquid Molar concentration Iron ion ratio Precipitated or non-acidic ion concentration ──────── (Molar concentration ────────────── Degree (Millimo A solution AB / molar concentration) Reaction time / litre after mixing 2 24 48 96 192 l) ──────────────────────── ──────────── 1.2 0.6 6 None None None None None 0.4654 0.8 0.4 4 None None None None None 0.2630 0.4 0.2 2 Yes None None None 0.0988 0.2 0.1 1 Yes Yes Yes None None 0.0005 0.1 0.05 0.5 Yes Yes Yes Yes Yes 0.0007 0.05 0.025 0.25 Yes Yes Yes Yes Yes 0.0004 ───────────────────────── ───────────

[0019]

[Table 5] At a reaction temperature of 37 ° C ─────────────────────────────── Minimum bicarbonate Bicarbonate ion Ion / mole Concentration Iron ion ratio Presence or absence of precipitation モ ル (Molar concentration 溶液 A solution AB / Molar concentration) Reaction time After mixing 2 24 48 96 192 ─────────────────────────────── 1.2 0.8 6 None None None None None 0.8 0.4 4 None None None None None 0.4 0.2 2 Yes Yes Yes Yes Yes 0.2 0.1 1 Yes Yes Yes Yes Yes 0.1 0.05 0.5 Yes Yes Yes Yes Yes 0.05 0.025 0.25 Yes Yes Yes Yes Yes Yes ───────────────── ──────────────

As described above, when the reaction is carried out at a low temperature for a long time, the iron-Lf complex of the present invention can be formed even when the carbonate ion and bicarbonate ion / iron ion ratio is 1. But,
If the concentration is lower than this, it cannot be produced even if the reaction is carried out for a long time. Further, when the concentration of carbonate ion and bicarbonate ion in the ultrafiltration membrane permeate after the reaction for 192 hours is the minimum bicarbonate ion / iron ion ratio is 1 or less, the concentration is the same as the concentration of deionized water. It is considered that all the bicarbonate in the solution was bound to the iron-Lf complex. Further, when the reaction is carried out at 37 ° C., the iron-Lf complex of the present invention cannot be formed if the ratio of carbonate ion and bicarbonate ion / iron ion is 2 or less.

From these results, it is considered that the iron-Lf complex of the present invention contains one or more molecules of carbonate and / or bicarbonate per bound iron. This iron-
It has also been found that increasing the concentration of carbonate ions and / or bicarbonate ions is effective in increasing the reaction rate of Lf complex formation. From this example, it can be seen that the i) molar concentration of iron ions in the B solution is the same as the ii) molar concentration of carbonate ions and bicarbonate ions in the solution (reaction solution) in which ii) the A solution and a part or all of the B solution are mixed. Even if it did, it became clear that an iron-Lf complex could be formed.
However, under these conditions, it takes an extremely long time to manufacture, and is not practical.

[0022]

Test Example 4 The number of moles of iron atoms and carbon atoms was further analyzed by elemental analysis to show the features of the iron-Lf complex of the present invention. Bovine Lf (Toyobo Co., Ltd.) with a purity of 99% purified by a monoclonal antibody is prepared by using a dialysis membrane (spectrometer) with a molecular weight of 10,000 cuts against a 0.1 mol / l citric acid solution containing 0.1% ethylenediaminetetraacetic acid (ultra pure water). (Pore Co., Ltd.) for 3 days, iron removal, dialysis against ultrapure water for another 4 days, complete removal of iron, and lyophilization. 10 ml of a pH 8.3 solution containing 1 mol / l sodium bicarbonate (solution A), 2 ml of a solution containing 300 micromoles of ferric chloride
(B1 solution), 8 ml of a solution containing 2.5 micromoles of Lf (B2 solution) was prepared. The B1 solution was mixed with the B2 solution (B solution), and 10 ml of the B solution was added to the A solution to prepare an iron-Lf complex. Similarly, B1
Iron-L prepared with 1.75 mmol ferric chloride in solution
The composite was designated as sample 2. In addition, the untreated Lf
Is a sample 3, and samples 1, 2 and 3 are
It was dialyzed with a dialysis membrane (Spectropore) having a molecular weight cut-off of 10,000 for 5 days and freeze-dried. Further, after powdering in an agate mortar, water was removed by a vacuum dryer at 45 ° C. for 48 hours. The obtained sample was analyzed for the iron content by an atomic absorption spectrometer (ICP) and the carbon content by a CHN elemental analyzer.

[0023]

[Table 6] Δ Sample ΔC ¹⁾ Fe ²⁾ ΔC / Fe (mol) (mol) ( Mol) ──────────────────────────── 1 60 117 0.5 2 71 706 0.1 ──────────── ──────────────── ¹⁾ (Total number of moles of carbon atoms per mole of sample) − (Lf
(Sample 3) Total number of moles of carbon atoms per mole ²⁾ Total number of moles of iron atoms in sample

From the results of the above elemental analysis, the iron-
It became clear that more carbon atoms and iron atoms were bonded to the Lf complex than usual Lf. Also, in this experimental system, the increase in carbon atoms was considered only to be supplied from baking soda, and this test example showed that carbonate ions or bicarbonate ions were bonded to iron atoms. . Since the ratio of carbon atoms to iron molecules was 0.1 or more, it was found that carbonate ions or bicarbonate ions were bonded to iron atoms at a ratio of 0.1 molecules or more. Further, in Test Example 3, it was considered that one molecule of bicarbonate was bonded to one molecule of iron, so it is considered that the bonding ratio of iron and carbon atoms was changed depending on the production conditions. In any case, it is clear from this test example that carbonate and / or bicarbonate ions and iron ions are bound to the iron-Lf complex. In sample 3, no iron atom was detected, and it was confirmed that almost all free iron was removed by dialysis.

[0025]

[Test Example 5] In JP-A-7-17875, after adding an iron salt to an Lf solution, increasing the pH by adding an alkali, and then removing free iron from the solution, the solution is evaporated, and the Lf solution is evaporated.
Per gram of Lf that comes out as a powder
A method is disclosed for producing iron / Lf powder that holds 2.1 grams or more of iron in a stable state. In addition, JP-A-4-
According to the publication No. 141067, at least 1.5
A production method is disclosed in which an iron compound corresponding to iron in a proportion of milligrams or more is added, and Lf is reacted with iron to prepare an iron / Lf solution. In this test example, the difference in structure between these iron / Lf solutions and the iron-Lf composite of the present invention is shown in test examples.

The Lf used was the one that had been deironed as shown in Test Example 4. The iron-Lf composite of the present invention was prepared in the same manner as in Sample 1 of Example 4, and used as Sample 1. JP
The iron / Lf solution disclosed in JP-A-7-17875 was prepared by adding 100 μmol of ferric chloride to 20 ml of a solution containing 2.5 μmol of Lf. Further, a 1-mol aqueous solution of sodium hydroxide was slowly added dropwise to this solution little by little, and the solution was adjusted to pH 9 to prepare Sample 2. The iron / Lf solution disclosed in JP-A-4-141067 was prepared as Sample 3 by adding 300 μmol of ferrous sulfate heptahydrate as iron to 20 ml of a solution containing 2.5 μmol of Lf. Further, as a control, Lf from which iron was removed was prepared in the same manner as in Example 4 to obtain Sample 4. The obtained four samples were desalted, freeze-dried, and vacuum-dried in the same manner as in Test Example 4, and then iron atoms were analyzed by an atomic absorption spectrometer, and nitrogen, carbon and hydrogen atoms were analyzed by a CHN elemental analyzer.

[0027]

[Table 7] Δ Sample ΔC ¹⁾ ΔH ²⁾ ΔN ³⁾ Fe ⁴⁾ (mol) (Mol) (mol) (mol) １ 160 303 0 117 2 3 23 0 193 3- 10 -8 1 20 ───────────────────────────── ¹⁾ (Total number of moles of carbon atoms per mole of sample)-( Lf
(Sample 4) Total number of moles of carbon atoms per mole) ²⁾ (Total number of moles of hydrogen atoms per mole of sample) − (Lf
(Sample 4) Total number of moles of hydrogen atoms per mole ³⁾ (Total moles of nitrogen atoms per mole of sample)-(Lf
(Sample 4) Total number of moles of nitrogen atoms per mole ⁴⁾ (Total moles of iron atoms per mole)

From the above results, the iron-Lf complex of the present invention (Sample 1) has carbon atoms, hydrogen atoms, and iron atoms bonded in a much larger amount than ordinary Lf as compared with ordinary Lf. You can see that there is. From this, the iron-L of the present invention
It is clear that the f-complex forms a structure different from ordinary Lf. On the other hand, in the iron / Lf solution (sample 2) disclosed in Japanese Patent Application Laid-Open No. 7-17875, since hydrogen atoms affect the effect of bound water and the like, slight variations are observed, but both carbon atoms, hydrogen atoms, and nitrogen atoms are affected. Since it is the same as ordinary Lf and only the iron atoms are increased, it is understood that iron exists in a state different from that of the iron-Lf complex of the present invention. Further, iron / L disclosed in JP-A-4-141067 is disclosed.
Similarly, in the f solution, carbon atoms, hydrogen atoms, and nitrogen atoms are the same as ordinary Lf, and it can be seen that only iron atoms are slightly increased. From the above, structurally, the iron-Lf composite of the present invention is disclosed in JP-A-7-17875 and JP-A-4-14.
It is clear that the iron / Lf solution of JP-A-1067 is completely different in structure.

It is conventionally known that, when Lf, iron and bicarbonate are mixed, iron-saturated Lf is generally formed. Lf is composed of two regions, a region called an N rope and a similar region called a C rope. Iron binds one molecule to each of these two regions. That is, in the N rope, Asp60, Tyr192, Tyr192, His253
One molecule of iron ion binds to the four amino acid residues, and one CO ₃ ^2- bond to this iron. Similar iron binding sites on the C rope are Asp395, Tyr435, Tyr528, Hi
s597 (BF Anderson et al., J. Mol. Biol. 20
9: 711-734, 1989). Therefore, the normal iron-saturated Lf means that 2 mol of iron and 2 mol of CO ₃ ^2- per mol of Lf.
Molar bonds. Further, the stability of the iron-saturated Lf is slightly higher than that of the Lf to which iron is not bound, but precipitates when heated at 65 ° C. or more. However, the iron according to the present invention
The Lf complex has much larger amounts of iron and carbonic acid and / or bicarbonate than so-called iron-saturated Lf.

In Japanese Patent Application Laid-Open No. 4-141067, L
It has been shown that when a large amount of iron is added to f, iron binds to Lf and becomes non-free, stabilizing the iron. However, adding a large amount of iron imparts heat resistance to Lf. Is not shown at all. Further, the publication does not disclose any necessity of carbonate ion or bicarbonate ion or use of carbonic acid / carbonate / bicarbonate, and carbonic acid / carbonate / bicarbonate is used in all Examples. not being used. Further, from Test Example 5, the number of iron atoms, carbon atoms, and hydrogen atoms is clearly different from the iron-Lf complex of the present invention.
Therefore, it is completely different from the iron-Lf complex according to the present invention.

The iron-Lf complex of the present invention contains carbonic acid and / or
Alternatively, the amount of bicarbonate bond and the amount of iron bond are much larger than the normal iron-saturated Lf in which two molecules of iron are bonded. Carbonate and / or bicarbonate and iron have a higher order structure of Lf (3
(Fourth, fourth) changes, and carbonic acid and / or bicarbonate, and iron are considered to surround Lf. In fact, the anti-Lf that normally recognizes iron-saturated Lf
The degree of recognition of the iron-Lf complex of the present invention by the antibody depends on the binding of carbonic acid and / or bicarbonate and iron to Lf.
It is getting lower. The situation is shown below.

[0032]

Test Example 6 Lf was used by dialyzing the Lf solution against a citrate buffer containing 0.1% ethylenediaminetetraacetic acid, removing iron, dialyzing against water, and freeze-dried. 1
1 liter of a solution of pH 8.3 containing mol / l of sodium bicarbonate (solution A)
To prepare a solution containing 480 mmol of 0.2 L (B1 solution) and a solution containing 1 mmol of Lf 0.8 L (B2 solution). After mixing the B1 solution and the B2 solution,
Diluted 00 times with deionized water (B solution). One liter of the B solution was added to the A solution to prepare Lf to which iron was bound. This solution was serially diluted with the simulated buffer. On the other hand, an ELISA plate was coated with an anti-bovine Lf antibody and then blocked with Block Ace (available from Dainippon Pharmaceutical Co., Ltd.).
After the Lf solution prepared above was added and reacted at room temperature for 1 hour, an anti-bovine Lf antibody labeled with peroxidase was reacted. After washing well, add ABTS substrate, 405nm
Was measured for absorbance. Thus, iron is 2 to Lf
When the molecules are bonded, they are recognized in the same manner as in the absence of iron, but are not recognized by the antibody as the amount of iron increases. Table 8 shows the results. The recognition rate in the table refers to the Lf contained in the solution of Lf to which iron is bound.
It refers to the ratio of the amount of Lf quantified by this measurement method to the amount.

[0033]

[Table 8] 認識 Recognition rate (%) with iron (mol / mol Lf) antibody ──── ─────────────────────── 0 1 0 0 2 1 0 0 1 5 9 0 3 0 8 6 6 0 8 2 9 0 6 0 1 2 0 3 1 1 5 0 1 9 2 4 0 1 4 4 8 0 1 4 ───────────────────────────

Further, the iron-Lf composite of the present invention is disclosed in
It is also different from iron / Lf of JP-A-6-239900 or JP-A-4-41067 in terms of pH stability. This is shown in the next test example.

Test Example 7 Preparation of the iron-Lf composite of the present invention was carried out in Test Example 6.
The same was done. The iron-Lf conjugate disclosed in JP-A-6-239900 is prepared by adding ferric chloride to an aqueous solution having an Lf of 33.75 micromoles / liter so that iron becomes 0.5 to 16.2 mmol / liter as iron and adjusting the pH to 6.2 with sodium bicarbonate. It was prepared by adjustment. The iron / Lf solution disclosed in JP-A-4-141067 was prepared by dissolving ferrous sulfate heptahydrate as iron in an aqueous solution in which Lf was dissolved to 12.5 micromol / deciliter in the form of iron at 187 to 6000 micromol / deciliter. . At this time, carbonic acid / carbonate / bicarbonate which generates carbonate ion and / or bicarbonate ion is not used. Each sample prepared by the above three methods was desalted and concentrated using an ultrafiltration membrane with a molecular weight of 5,000, and a simulated buffer (pH 6.4 or 7.2)
Then, the solution was diluted so as to have an Lf of 125 micromol / liter. The pH was adjusted to 6.5 and 7.3, respectively, sealed in a test tube with a screw hole, heated at 90 ° C for 10 minutes, allowed to cool to room temperature, centrifuged at 3,000 rpm for 10 minutes, and the Lf content in the supernatant was measured. Was measured. This measurement was performed using BioRad
The Protein Assay Kit manufactured by the company was used. Table 9 shows the ratio of Lf remaining in the supernatant.

[0035]

Table 9: JP-A-4-141067 JP-A-6-239900 The present invention Fe / Lf molar ratio ───────── ───────── ──────── pH6.5 pH7.3 pH6.5 pH7.3 pH6.5 pH7. 3 ──────────────────────────────────── 15 5% 6% 80% 43% 100% 100% 30 7 6 84 40 100 100 60 4 3 97 54 100 100 120 6 1 98 38 100 100 150 5 4 81 27 100 100 240 9 2 79 21 100 100 480 2 6 80 13 98 89 ──────── ────────────────────────────

As described above, according to the present invention, pH 6.5 or
Although heat resistance was shown in any of 7.3, JP-A-6-239
No. 900 showed heat resistance at pH 6.5, but did not show heat resistance at pH 7.3. Also, in JP-A-4-141067,
Neither pH 6.5 nor 7.3 showed heat resistance.

[0037]

Test Example 8 It is known that in a region where the salt concentration is low, even Lf has heat resistance (Japanese Patent Laid-Open No. 4-8269). The following shows that only the iron-Lf complex of the present invention has heat resistance in a region where the salt concentration is high. Production of the iron-Lf composite of the present invention was performed in the same manner as in Test Example 6. Preparation of the iron-Lf conjugate disclosed in JP-A-6-239900 and the iron / Lf solution described in JP-A-4-41067 were carried out in the same manner as in Test Example 7. Also, JP-A-7-17
No. 875 iron / Lf solutions were prepared for two methods, A and B. In the method A, ferric chloride was added to an aqueous solution in which Lf was dissolved so as to have a concentration of 12.5 micromol / deciliter so as to have a concentration of 0.5 to 16.2 mmol / l as iron, and the pH was adjusted to 9.0 with 1N sodium hydroxide. Produced. In the method B, Lf was dissolved in a 50 mmol / liter ammonium oxalate solution (pH 8.0) so that the concentration became 12.5 micromol / deciliter, and ferric chloride was dissolved.
Add 0.5-16.2 mmol / L and add 1N
It was prepared by adjusting the pH to 9.0 with sodium hydroxide. Each sample prepared by the above five methods was desalted and concentrated with an ultrafiltration membrane (Advantech) with a molecular weight cutoff of 10,000, and diluted with ultrapure water to a Lf of 250 μmol / L. did. The electric conductivity at this time was 0.2 millisiemens / cm. Further dilute it 4 times with a simulated buffer (pH 7.0) to further increase the salt concentration.
Salt was added so that the concentration became 30 mmol / liter. After sealing in a test tube with a screw opening, the mixture was heated at 90 ° C. for 10 minutes, and the precipitation of Lf was observed. A sample with no precipitation was rated 0, a sample with a slight precipitation was rated 1, a sample with a large amount of precipitation, and a sample 2 which had already precipitated during the preparation of the iron / Lf solution.

[0038]

Table 10 ─────────────────────────────────── Fe / Lf ratio 7-17875 JP- A- 6-239900 Method A of the present invention Method B Method 30 2 2 0 2 0 2 0 0 1 2 2 2 0 0 150 2 2 2 1 0 240 2 2 2 2 0 ────── ─────────────────────────────

It has already been disclosed that Lf has heat resistance when its ionic strength is low (JP-A-4-8269). Among these, the electric conductivity range in which the Lf aqueous solution or the iron-bonded Lf can be sterilized by heating is disclosed by the following formula. LogΩ ≦ 2.96 / pH + 0.64 (pH <5) LogΩ ≦ 29.37 / pH-4.62 (5 ≦ pH ≦ 7.9) LogΩ ≦ -0.917 (pH> 7.9) where Ω is the electrical conductivity (milliSiemens / cm) Show. For example, in this test example, the pH after desalination was about 7.0.
Therefore, when the electric conductivity range in which normal Lf can be heat-sterilized at pH 7.0 is calculated, the range is as follows.
From the formula, LogΩ ≦ 29.37 / 7-4.62 ≦ -0.42 From this Ω ≦ 10 ^-4.62 ≦ 0.38

That is, it has already been clarified that sterilization is possible if the electric conductivity is 0.38 millisiemens / cm or less. Therefore, Lf can be heat-sterilized by ordinary desalination treatment. Since each sample had a different preparation method, the samples were all desalted once, the salt concentration was increased again with sodium chloride, and the heat resistance test was performed under the same conditions. In the desalination treatment performed this time, desalination was performed to 0.2 millisiemens / centimeter, and 0.03 mol / liter of salt was added, and a heat resistance test was performed. The electric conductivity at this time was 0.53 milliSiemens / cm.
This value is a higher value, and is a region in which heat resistance is not recognized by ordinary Lf. From Table 10, it can be seen that the iron-
No precipitation was observed in the Lf complex, indicating that it had a higher thermal stability to heat than ordinary Lf.
Japanese Patent Application Laid-Open No. 6-239900 also showed high thermal stability different from ordinary Lf, but became unstable where the Fe / Lf ratio was high. In JP-A-4-141067 and JP-A-7-17875, precipitation was observed, and it was shown that they had only the same heat resistance as ordinary Lf. This indicates that the iron-Lf complex of the present invention has a different structure from ordinary Lf and other disclosed iron / Lf solutions and exhibits high heat resistance.

Test Example 9 shows the heat resistance of the composition of the present invention at a higher electric conductivity.

Test Example 9 An iron-Lf composite was prepared in the same manner as in Test Example 6. However, the iron content of the B1 solution was up to 1000 mmol. This solution was desalted and concentrated using an ultrafiltration membrane having a molecular weight of 5,000, and then diluted with a simulated buffer solution to have an Lf content of 625 micromol / liter. The simulated buffer in this case is glycine-hydrochloric acid at pH 2.0 to 3.5, pH 3.5 to 6.
0 is acetic acid, pH 6.0-7.8 is imidazole-hydrochloric acid, pH 7.8
9.3 used boric acid-potassium chloride-sodium hydroxide as a buffer. The electric conductivity was adjusted using sodium chloride. After sealing this in a test tube with a screw hole, 90
After heating at ℃ for 10 minutes and naturally cooling to room temperature, 3,000r
After centrifugation at pm for 10 minutes, the Lf content in the supernatant was measured. Note that a Protein Assay Kit manufactured by BioRad was used for this measurement. Tables 11 and 1 show the percentage of Lf remaining in the supernatant.
It is shown in FIG.

[0042]

[Table 11] When the electric conductivity is 5 millisiemens / cm Fe / Lf pH molar ratio 2.0 2.1 2.5 3.0 4.0 5.0 6.0 7.0 8.8 9.0 9.2 ──── ────────────────────────────── 10 59 58 57 52 50 46 33 38 31 32 34 (%) 15 50 73 74 72 71 75 76 80 77 72 45 30 46 82 91 95 96 97 99 100 100 100 36 60 51 95 93 97 98 100 100 100 100 100 43 120 54 97 100 100 100 100 100 100 100 100 39 240 32 99 100 100 100 100 100 99 99 97 40 480 21 92 93 96 100 100 100 98 96 93 47 1000 20 76 77 78 79 78 95 94 92 92 39 1200 19 32 28 31 33 27 29 30 24 33 32 ───────── ─────────────────────────

[0043]

[Table 12] When the electrical conductivity is 150 millisiemens / cm Fe / Lf pH molar ratio 2.0 2.1 2.5 3.0 4.0 5.0 6.0 7.0 8.8 9.0 9.2 ─── ─────────────────────────────── 10 51 50 52 53 49 46 37 34 31 32 31 (%) 15 44 70 71 72 70 73 72 73 72 64 40 30 41 73 82 85 85 85 87 88 89 87 34 60 45 84 82 84 85 87 89 88 90 89 36 120 47 87 89 90 90 90 90 92 91 88 34 240 28 88 89 90 90 90 90 89 90 87 35 480 20 79 82 83 81 83 86 85 82 80 41 1000 18 66 68 68 70 69 84 83 80 79 34 1200 19 26 26 28 30 27 26 27 25 29 26 ──────── ──────────────────────────

As described above, the amount of iron was 15 per Lf1 molecule.
Heat resistance was not obtained unless the number of molecules was not less than 1000 and not more than 1,000, and within this range, heat resistance was confirmed in the range of pH 2.1 to 9.0. More specifically, iron is 15 per Lf1 molecule.
Thirty molecules have higher heat resistance than molecules, and 60 molecules have more heat resistance than 30 molecules, and 120 molecules have higher heat resistance than 60 molecules. Also, iron is 1000 per Lf1 molecule.
480 molecules have higher heat resistance than molecules, and 240 molecules have higher heat resistance than 480 molecules. When 1000 molecules of iron are bound to one Lf molecule, the heat resistance is higher when the pH exceeds 5 than when the iron is 5 or less. The iron-Lf conjugate prepared by the method of JP-A-6-239900 precipitates because iron cannot bind to Lf more than 720 molecules, but in the present invention, it can bind 1000 molecules of iron. From this point, the iron-Lf of the present invention
Composite and iron-L produced by the method of JP-A-6-239900
The f-conjugates can be said to be different. In Japanese Patent Application Laid-Open No. 4-8269, when sterilizing a beverage containing Lf, the final pH of the product is related to the electrical conductivity Ω, and the relation is log Ω (milli-Siemens / cm)> (2.96 / p
H) +0.64 (pH <5) log Ω> (29.37 / pH) −4.62 (5 ≦ pH ≦ 7.9) log Ω> −0.917 (pH> 7.9) It is said that it will undergo denaturation so that it can no longer bind iron.

When a product containing the iron-Lf complex obtained according to the present invention is produced, the product is 150 milliSiemens / centimeter and has a very stable heat resistance even when the pH is in the range of 2.1 to 9.0. Since it is an iron-Lf complex, even if the relationship between Ω and pH is other than the relationship described above, there is no problem in production.

Next, the present invention and JP-A-6-239900,
The difference in storage stability between 7-17875 and JP-A-4-41067 is shown.

Test Example 10 An iron-Lf complex, an iron-Lf conjugate and an iron / Lf solution were prepared in the same manner as in Test Examples 7, 8, and 9, and desalted in the same manner as in Test Example 9. The electric conductivity was adjusted to be Milli-Siemens. Each centrifuged sample was sealed and heated at 90 ° C. for 10 minutes, and then stored at room temperature (37 ° C.) for one month. Thereafter, the amount of the precipitate was visually determined.
0: no precipitation was observed, 1: slight precipitation was observed, and 2: heavy precipitation was observed. In addition, JP-A-7-17
For 875, the storage period was 3 weeks. Test example 7
Table 13 shows the results of iron-Lf conjugate and iron / Lf solution of
Table 14 shows the results of the iron-Lf composite of Test Example 9.

[0047]

[Table 13]

[0048]

[Table 14] {Fe / Lf pH mol}比 Ratio 2.0 2.1 2.5 3.0 4.0 5.0 6.0 7.0 8.8 9.0 9.2 ────────────────── ─────────────── 10 2 2 2 2 2 1 1 1 2 2 2 15 2 1 0 0 0 0 0 0 0 0 2 30 2 1 0 0 0 0 0 0 0 0 2 60 2 0 0 0 0 0 0 0 0 0 2 120 2 0 0 0 0 0 0 0 0 0 2 240 2 0 0 0 0 0 0 0 0 0 2 480 2 0 0 0 0 0 0 0 0 0 2 1000 2 1 1 1 1 0 0 0 0 0 2 1200 2 2 2 2 2 2 2 2 2 2 2 ─────────────────────────── ──────

Tables 13 and 14 show that the iron-Lf of the present invention
The complex had high storage stability at room temperature even after being subjected to a heat stress of 90 ° C. for 10 minutes in a pH range of 2.1 to 9.0. Table 13 shows that the iron-Lf composite of the present invention has storage stability not found in the Lf disclosed in other patents. JP-A-6-239900 Fe / L at pH 6.5
It can be seen that the composition is stable when the f molar ratio is 150 or less, but has no storage stability at pH 7.3. Also, JP-A-4-141067
No. Lf solution at pH 6.5 and 7.3 already lacked heat resistance and thus naturally lacked storage stability. Also, in JP-A-7-17875, storage stability was not recognized in both the methods A and B.

When manufacturing a product containing Lf obtained according to the present invention, the manufacturing process is not particularly limited. Sterilization is processed according to the usual process,
Heat sterilize or sterilize. Heat treatment is pasteurization at 65 ° C for 30 minutes, 120 ° C for 2-3 seconds, 140-150 ° C, 3-5
Seconds or retort treatments are also possible. When dried, an Lf-containing powder product can be obtained. Although freeze-drying may be used, spray drying with low drying cost is suitable for large-scale treatment. For the purpose of redissolving, mixing with skim milk, whey, casein, gelatin, sucrose, starch, etc. improves the solubility. Since the Lf-containing product thus obtained contains iron,
Particularly suitable for foods, feeds and pharmaceuticals for the purpose of preventing or treating anemia. In particular, since the astringent taste of iron is completely suppressed, it is excellent as an iron material for iron-fortified foods and orally administered pharmaceuticals. Further, it has a property of preventing the effect of promoting the generation of peroxide by iron, and thus has a high utility value as an iron material for easily oxidized foods such as fat.

[0051]

Test Example 11 An iron-Lf composite was prepared in the same manner as in Test Example 7,
An iron-Lf conjugate and an iron / Lf solution were prepared. However,
The final concentration of Lf was 13.2 micromol / liter, and the final concentration of the added iron agent was adjusted to be 1.98 mmol / liter in terms of iron. As the iron agent, ferric chloride was used for the iron-Lf complex of the present invention and the iron-Lf conjugate of JP-A-6-239900, and ferrous sulfate was used for the Lf solution of JP-A-4-141067. It was used. As a control, an aqueous solution of 1.98 mmol / L ferric chloride, an aqueous solution of 1.98 mmol / L ferrous sulfate, and a 13.2 μmol / L Lf aqueous solution containing 1.98 mmol / L ferric chloride were prepared. Sample No. 7 was prepared by simultaneously adding 1.98 mmol / L ferric chloride and 13.2 μmol / L Lf to an aqueous solution of sodium bicarbonate, and was manufactured without following the manufacturing method of the present invention. Each of the obtained samples was desalted by adding 1 ml thereof to a mol cut (Millipore) having a molecular weight of 10,000. When the retentate amount of each sample reached 0.2 ml, 1 ml of water was added, and a total of 6 ml of water was added. The iron content of the obtained retentate and permeate was measured by an atomic absorption spectrometer (ICP). In addition, all the shown molar concentrations were shown as final concentrations. Further, the retentate iron remaining rate was determined by the following equation. Retained iron residual rate (%) = {Retained iron total amount}
(Total amount of retentate iron + Total amount of permeate iron)｝ × 10
0

Furthermore, the astringent taste of iron was tested before treating the prepared samples with an ultrafiltration membrane. Table 1 shows the results
It is shown in FIG. Sample 1: Iron-Lf complex of the present invention Sample 2: Iron-Lf conjugate of JP-A-6-239900 Sample 3: 1.98 mmol / L Ferric chloride-containing 13.2 μmol / L Lf aqueous solution Sample 4: JP-A-Hei. 4-141067 Lf solution Sample 5: 1.98 mmol / L ferric chloride aqueous solution Sample 6: 1.98 mmol / L ferrous sulfate aqueous solution Sample 7: 1.98 mmol / L ferric chloride and 13.2 μmol / L Lf Aqueous sodium bicarbonate solution

[0053]

[Table 15] ─────────────────────────────── Sample No. Retained iron residual rate (%) Astringency ──── １ 1 99.3 None 2 98.1 None 3 25.3 Yes 4 10.9 Yes 5 6.2 Yes 6 3.9 Yes R 7 23.2 Yes

In Samples 5 and 6, all of the iron migrated to permeate, indicating that the iron agent used in the aqueous solution permeated the 10,000-cut membrane. on the other hand,
In Samples 1 and 2, all iron remained in the retentate, and it was found that the iron bound to Lf and did not pass through the membrane with a molecular weight of 10,000 cut. Sample 3 contains iron and L
f shows the state before the mixture is added to the aqueous sodium bicarbonate solution, in which a large amount of iron is present in a free state, and iron has been transferred to permeate. Furthermore, Sample 7 shows that a large amount of free iron is present simply by simultaneously contacting ferric chloride, Lf and sodium bicarbonate.

From the above results, it was confirmed again that the preparation of the iron-Lf complex of the present invention requires an aqueous sodium bicarbonate solution, and that the preparation procedure is also important. In addition, in sample 4, iron was almost completely changed to permeate, and it was found that iron was in a free state. Regarding the astringent taste of iron (iron taste), samples 1 and 2
Was not observed in other samples, but was observed in other samples.

Next, the results of taste are shown.

[Test Example 12] Similar to Test Example 7, various iron / Lf mixed solutions prepared by the three methods were desalted and concentrated using an ultrafiltration membrane with a molecular weight cut-off of 5,000, and then simulated buffer solution (pH 6.8). It was diluted with distilled water to be 13 mg / 100 ml and 26 mg / 100 ml as iron ions.
The thus obtained sample was subjected to the following sensory evaluation test. Panelists of 5 males and 5 females were asked to judge whether or not they felt the astringent taste of each sample using the simulated buffer as a control. The panelists were blindfolded so as not to give any external judgment factors. The test for one sample was tasted in the order of the control and the sample, and after the evaluation of one sample, a test for evaluating the next sample was performed at least one day apart. In addition, the order of sample evaluation was randomized for each panelist in order to eliminate the daily deviation of sample evaluation. As a result, Table 16 shows the number of panelists who felt astringent taste among the ten panelists.

[0057]

Table 16】 JP-A-4-141067 JP-A-6-239900 The present invention Fe / Lf molar ratio ───────── ───────── 濃度 Iron concentration (mg / 100 ml) 13 26 13 26 13 26 ── ────────────────────────────────── 15 10 10 0 0 0 0 30 10 10 0 0 0 0 60 10 10 0 0 0 0 150 10 10 0 3 0 0 240 10 10 1 4 0 0 480 10 10 1 6 0 0 ────────────────── ──────────────────

As described above, it has been clarified that the present invention has an excellent masking effect that does not cause any astringent taste of iron. Also, in JP-A-6-239900, the iron concentration is 26 mg / 100 ml and the Fe / Lf molar ratio is 150 mg.
In the case of Japanese Patent Application Laid-Open No. 4-141067, all 10 panelists recognized the astringent taste in all cases.

[0059]

[Test Example 13] In the same manner as in Test Example 9, the iron-Lf complex was desalted and concentrated using an ultrafiltration membrane having a molecular weight of 5,000, and the concentration of the iron ion was set to 26 mg / 100 ml in a simulated buffer solution. It was diluted and sterilized at 90 ° C. for 10 minutes. The sample thus obtained was subjected to a sensory evaluation test in the same manner as in Test Example 12.

[0060]

[Table 17] {Fe / Lf pH mol}比 Ratio 2.0 2.1 2.5 3.0 4.0 5.0 6.0 7.0 8.8 9.0 9.2 ────────────────── ─────────────── 10 10 9 8 8 7 6 6 5 6 8 10 15 10 3 2 2 1 0 0 0 0 1 10 30 10 1 0 0 0 0 0 0 0 0 10 60 10 0 0 0 0 0 0 0 0 0 10 120 10 0 0 0 0 0 0 0 0 0 10 240 10 0 0 0 0 0 0 0 0 0 10 480 10 2 1 1 1 1 0 0 0 0 10 1000 10 3 2 2 2 1 0 0 0 0 10 ──────────────────────────────────

As described above, the iron-Lf complex of the present invention has an environment surrounding pH 2.1 to pH 9.0 as long as 15 to 1,000 molecules of iron are bonded to one Lf molecule. Within this range, it had an effect of suppressing the astringent taste of iron. At this time, iron bound more than 480 molecules to one molecule of Lf, and several people felt a slight astringent taste in the range of pH 2.1 to pH 5.0, but when the iron content was 13 mg / 100 ml, 10 people felt iron. None of the panelists felt astringent taste.

[0062]

Example 1 1.2 mol of sodium bicarbonate and Lf (DMV
1 liter (A) containing 10 micromolar.
Solution 1 containing 1.5 mmol of ferric sulfate as iron ion
A liter (B) was made. B was added to A to prepare Lf to which iron was bound. This sample was desalted and concentrated using an ultrafiltration membrane having a molecular weight of 5,000, and diluted with a simulated buffer solution (pH 8.9) so that the iron concentration was 26 mg / 100 ml. After sealing this in a test tube with a screw hole, 90 ° C
After heating for 10 minutes and naturally cooling to room temperature,
Saved for months. Precipitation was judged with the naked eye, and was not observed at all. Further, when a sensory evaluation test was performed in the same manner as in Test Example 12, none of the ten panelists felt astringent taste.

[0063]

Example 2 One liter of a solution containing 0.05 mol of calcium carbonate and 1.2 mol of ammonium bicarbonate was adjusted to pH 7.8 with hydrochloric acid (solution A). Ferric sulfate as iron ion 1.5
A solution containing 0.2 microliter of a solution containing mmol (B1 solution) and a solution containing 10 micromol of transferrin (apo-type, high-purity, bovine plasma, Wako Pure Chemical Industries, Ltd.) were prepared as 0.8 liter (solution B2). After mixing the B1 solution and the B2 solution, a B1 / B2 mixed solution was added to the A solution to form iron-bound transferrin. The iron-transferrin binding solution was desalted and concentrated using an ultrafiltration membrane having a molecular weight of 5,000, and diluted with a simulated buffer solution (pH 6.8) to an iron concentration of 26 mg / 200 ml. This was sealed in a test tube with a screw hole, heated at 90 ° C. for 10 minutes, allowed to cool to room temperature, and then stored at 37 ° C. for one month. Precipitation was judged with the naked eye, and was not observed at all. Further, when a sensory evaluation test was performed in the same manner as in Test Example 12, panelists
None of the 10 felt astringent taste.

[0064]

Example 3 0.5 mol of sodium carbonate, potassium bicarbonate
One liter of a solution containing 0.7 mol was adjusted to pH 8.3 with acetic acid (solution A). 0.2 liter of a solution containing 1.5 mmol of iron (III) nitrate as iron ion (B1 solution), 0.8 liter of a solution containing 10 micromol of ovotransferrin (type IV, crude, egg white, ironless, Sigma) (B2 solution) It was created. After mixing the B1 solution and the B2 solution, the B1 / B2 mixed solution was added to the A solution to prepare iron-bound ovotransferrin. The iron-ovotransferrin binding solution was desalted and concentrated using an ultrafiltration membrane having a molecular weight of 5,000. This was diluted with a simulated buffer solution (pH 6.2) so that the iron concentration was 26 mg / 200 ml. This was sealed in a test tube with a screw hole, heated at 90 ° C. for 10 minutes, allowed to cool to room temperature, and then stored at 37 ° C. for one month. Precipitation was judged with the naked eye, and was not observed at all. Further, when a sensory evaluation test was performed in the same manner as in Test Example 12, one of ten panelists who felt astringent taste was one.
No names were found.

[0065]

Example 4 One liter of a solution containing 1.0 mol of sodium bicarbonate was prepared (solution A). 0.2 liter containing 1 mmol of ferric chloride as iron ion (B1 solution), 10 micromol of bovine lactoferrin (manufactured by ULN) 0.8
One liter (B2 solution) was prepared. After mixing B1 and B2, a B1 / B2 mixture was added to the A solution. To maintain the pH of the solution at 8.5, sodium bicarbonate was added as appropriate to make the iron-Lf complex. The obtained iron-Lf complex was heated at 90 ° C. for 10 minutes without desalting, and was naturally cooled to room temperature. No precipitate was observed in this state. Furthermore, it was stored at 37 ° C. for one month, and the precipitate was again judged with the naked eye. As a result, no precipitate was observed. Further, when a sensory evaluation test was performed in the same manner as in Test Example 12, none of the 10 panelists felt astringent taste.

[0066]

Example 5 One liter of a solution containing 0.8 mol of sodium bicarbonate was prepared (Solution A). 0.2 liter (B1 solution) containing 1 mmol of ferric sulfate as iron ion, 10 micromol of bovine lactoferrin (manufactured by ULN) 0.8
One liter (B2 solution) was prepared. After mixing B1 and B2, a B1 / B2 mixture was added to the A solution to prepare an iron-Lf complex.
The obtained iron-Lf complex was heated at 90 ° C. for 10 minutes without desalting, and was naturally cooled to room temperature. No precipitate was observed in this state. Furthermore, it was stored at 37 ° C. for one month, and the precipitate was again judged with the naked eye. As a result, no precipitate was observed. Further, when a sensory evaluation test was performed in the same manner as in Test Example 12, none of the 10 panelists felt astringent taste.

[0067]

Example 6 A solution was prepared by lowering the pH of a 1-liter solution containing 1.0 mol of sodium carbonate to 8.0 with 2N hydrochloric acid.
Solution containing 1 mmol of ferric sulfate as iron ion 0.2
Liter (B1 solution), bovine lactoferrin (ULN) 10
0.8 liter (B2 solution) containing micromolar was prepared. After mixing B1 and B2, a B1 / B2 mixture was added to the A solution to prepare an iron-Lf complex. The obtained iron-Lf complex was heated at 90 ° C. for 10 minutes without desalting, and was naturally cooled to room temperature. No precipitate was observed in this state. In addition, 37 ℃
And stored again for one month, and again the precipitate was visually judged, and no precipitate was observed. Further, when a sensory evaluation test was performed in the same manner as in Test Example 12, none of the 10 panelists felt astringent taste.

[0068]

Example 7 Eight liters of a solution containing 24 mol of sodium bicarbonate (insoluble salts precipitated) were prepared (A
solution). 2 liters of a solution containing 20 mmol of ferric chloride as iron ion (B1 solution), solution 8 containing 100 micromoles of lactoferrin (Tatsua Biologics)
One liter (B2 solution) was made. After mixing the B1 solution and the B2 solution, the B1 /
B2 mixture was added to form iron-bound lactoferrin. The solution was hydrodesalted with an ultrafiltration membrane having a molecular weight of 5,000 and then concentrated to 1 liter with the same membrane. 20 liters of reduced skim milk (100 g of skim milk powder in 1 liter of water)
(Reduced in liters), the iron concentration was 26 mg / 200
It was added to make up milliliters. After stirring and mixing this,
The solution was sterilized at 120 ° C. for 2 seconds using a plate-type sterilizer, immediately cooled to 5 ° C., and then stored at a low temperature (10 ° C.) for 2 weeks. A portion was centrifuged at 3,000 rpm for 10 minutes, and the precipitate was visually determined. Further test examples
A sensory evaluation test was performed in the same manner as in 10. However,
As the control, reduced skim milk sterilized and stored under the same conditions was used. None of the 10 panelists felt abnormal in astringency, taste, or aroma.

[0069]

Example 8 Iron / Lf prepared, concentrated and desalted in Example 4
The mixed solution was added to 20 liters of raw milk with an iron concentration of 26 milligrams.
/ 200 ml. After homogenization, the mixture was sterilized at 150 ° C. for 4 seconds, immediately cooled to 4 ° C., and aseptically filled into 250 ml paper containers. After storage at 37 ° C. for 3 months, the mixture was centrifuged to examine the presence or absence of a precipitate, but no precipitate was observed. In addition, the number of E. coli and the number of general bacteria were both 0. In addition, browning might proceed during storage due to the presence of iron, but no browning was observed. Further, a sensory evaluation test was performed in the same manner as in Test Example 12. However, a control was prepared by sterilizing and storing raw milk under the same conditions. None of the 10 panelists felt abnormal in astringency, taste, or aroma.

[0070]

Example 9 Iron / Lf prepared, concentrated and desalted in Example 4
A physiological solution containing a complex solution (test group) or a ferrous sulfate solution (control group 1) containing 6.2 mg / 100 g of ascorbic acid and sodium ascorbate as vitamin C so that the iron concentration becomes 20 mg / 100 ml. A sample dissolved in a phosphate buffer (pH 7.2) and sterilized at 90 ° C. for 10 minutes was used as a test sample. As a control group 2, a physiological phosphate buffer to which vitamin C was added was also sterilized. Immediately after weaning, a 21-day-old Wistar female rat (Charles River Japan), weighing 45 to 50 grams, was treated with an iron-free diet (Oriental yeast, iron content 0.25).
(Milligram / 100 gram feed) for 2 weeks to produce anemic rats having a blood hemoglobin value of 7 g / 100 ml or less. Each group consisted of 4 rats, and the test sample was administered at a rate of 1 ml / day, 6
Weekly gavage (sonde) administration. 6 after test sample administration
On the week, blood was collected from the tail vein and the hemoglobin value was measured with an automatic blood cell counter (Toa Medical Electronics). Table 18 shows the results.

[0071]

[Table 18] ────────────────────────── Hemoglobin value (mean ± standard deviation) ─────────── ─────────────── Test group 15.2 ± 1.1 (g / 100 ml) Control group 1 12.9 ± 0.9 Control group 2 4.8 ± 0.3 ──────────── ──────────────

As described above, it was revealed that the iron-Lf complex of the present invention exhibited an anemia treatment effect, and that the effect was superior to that of ferrous sulfate, which is an inorganic iron.

[0073]

Example 10 Iron-L prepared, concentrated and desalted in Example 4
f The complex solution was added to raw milk to which ascorbic acid and sodium ascorbate were added as vitamin C in an amount of 30 mg / 200 ml as vitamin C, so that the iron concentration was 15 mg / 200 ml. It was filled up to 10 ml or less (test group). As a control, vitamin C enriched milk to which sodium iron citrate was added instead of the iron-Lf complex was used (control group). This was retort-sterilized at an F value of 4, stored at 37 ° C. for 2 weeks, and the remaining vitamin C content was quantified using a vitamin C measuring device (TOA Denshi).
The residual ratio of vitamin C was defined as a percentage obtained by dividing the measured value by the initial value. Table 19 shows the results.

[0074]

[Table 19]

As described above, it has been clarified that the iron-Lf complex has a lower degree of destruction of vitamin C than inorganic iron and is also effective as an iron material having a low ability to generate oxidation and peroxide.

Further, the test group was subjected to a sensory evaluation test in the same manner as in Test Example 12. However, as a control, a vitamin C enriched milk to which no iron was added was retorted under the same conditions. None of the 10 panelists felt abnormal in the astringent taste or the like.

[0077]

Example 11 One gram of Lf (manufactured by DMV) was dissolved in 0.2 mol / l acetate buffer (pH 4), and 26000 units of pepsin (Sigma) were added. The pH was adjusted to 7.5 with sodium. Add 200,000 units of trypsin (Sigma) to this, and add
Allowed to react for hours. It was confirmed by electrophoresis that the thus obtained decomposed Lf was mainly a decomposed product having a molecular weight of 50,000, 40,000 and 30,000. Sodium bicarbonate
One liter of a solution containing 1.3 mol (insoluble salt precipitated) was prepared (solution A). 0.2 liter of a solution containing 1.2 mmol of ferric chloride as iron ion (B1 solution) and 0.8 liter of a solution containing 10 micromol of lactoferrin as a lactoferrin before decomposition of the lactoferrin-decomposed product (B2 solution) were prepared. After mixing the B1 solution and the B2 solution, the B1 / B2 mixed solution was added to the A solution while stirring the A solution well, to prepare an iron-bound lactoferrin degradation product. This was sealed in a test tube with a screw hole, heated at 90 ° C. for 10 minutes, allowed to cool to room temperature, and then stored at 37 ° C. for one month. Precipitation was judged with the naked eye, and was not observed at all. Further, when a sensory evaluation test was conducted in the same manner as in Test Example 12, none of the 10 panelists felt astringent taste.

──────────────────────────────────────────────────続 き Continued on front page (58) Field surveyed (Int.Cl. ⁶ , DB name) C07K 14/79 A61K 38/16

Claims (6)

(57) [Claims] 1. A carbonic acid and / or bicarbonate-iron-lactoferrin complex having the following properties: 1) Each molecule of lactoferrin contains 15 to 1000 molecules of iron and 15 or more molecules of carbonic acid and / or bicarbonate. 2) No precipitation at room temperature at pH 2.1 to 9.0 at room temperature for at least one month. 3) No precipitation occurs even when heated. 4) No astringent taste unique to iron. 2. The carbon atom and the iron atom bound to the iron-lactoferrin complex, wherein ((total moles of carbon atoms per mole of the iron-lactoferrin complex according to claim 1)-( The complex according to claim 1, wherein the value of (total number of moles of carbon atoms per mole of lactoferrin not present)) / (total moles of iron atoms per mole of iron-lactoferrin complex according to claim 1) is 0.1 or more. . 3. Carbon dioxide, or ii) bicarbonate, or iii)
A solution containing carbonic acid and bicarbonate (solution A) is mixed with iv) a solution containing iron and v) lactoferrins (solution B) to obtain carbonic acid having properties shown in 1) to 4) below. And / or bicarbonate-iron-lactoferrin complex. However, at this time, the vi) iron ion molar concentration of the B solution is determined by vii) the molar concentration of carbonate ion and bicarbonate ion dissolved in a solution (reaction solution) in which the A solution and a part or all of the B solution are mixed. The molar concentration of ix) lactoferrins in the B solution is 1/15 to 1/1000 of the molar concentration of x) iron ion in the B solution. 1) Each molecule of lactoferrin contains 15 to 1000 molecules of iron and 15 or more molecules of carbonic acid and / or bicarbonate. 2) Prevents precipitation at room temperature at a pH of 2.1 to 9.0 for at least one month. ) No precipitation when heated. 4) No astringent taste unique to iron. 4. A method according to claim 1, wherein i) carbonic acid, or ii) bicarbonate, or iii).
Carbonic acid and bicarbonate, and iv) Carbonic acid obtained by mixing a solution containing lactoferrins (A solution) and v) a solution containing iron (B solution) and exhibiting the following properties 1) to 4): And / or bicarbonate-iron-lactoferrin complex. However, at this time, the vi) iron ion molar concentration of the B solution is determined by vii) the molar concentration of carbonate ion and bicarbonate ion dissolved in a solution (reaction solution) in which the A solution and a part or all of the B solution are mixed. Less than 1/3 of the concentration, ix) A
The molar concentration of lactoferrins in the solution is 1/15 to 1/1000 of the molar concentration of x) iron ions in the B solution. 1) Each molecule of lactoferrin contains 15 to 1000 molecules of iron and 15 or more molecules of carbonic acid and / or bicarbonate. 2) Prevents precipitation at room temperature at a pH of 2.1 to 9.0 for at least one month. ) No precipitation when heated. 4) No astringent taste unique to iron. 5. i) Carbonic acid, or ii) Bicarbonate, or iii)
Carbonic acid exhibiting the following properties 1) to 4), characterized by mixing a solution containing carbon dioxide and bicarbonate (A solution) with a solution containing iv) iron and v) lactoferrins (B solution). And / or a method for producing a bicarbonate-iron-lactoferrin complex. However, at this time, the vi) iron ion molar concentration of the B solution is determined by vii) the molar concentration of carbonate ion and bicarbonate ion dissolved in a solution (reaction solution) in which the A solution and a part or all of the B solution are mixed. Less than 1/3 of the concentration and B
Ix) of the solution B)
It is 1/15 to 1/1000 of the iron ion molar concentration. 1) 15 to 1000 iron molecules per lactoferrin molecule,
And at least 15 molecules of carbonic acid and / or bicarbonate; 2) no precipitation at room temperature for at least one month at pH 2.1 to 9.0; 3) no precipitation even when heated; 4) iron No unique astringent taste. 6. i) carbonic acid, or ii) bicarbonate, or iii)
It exhibits the following properties 1) to 4), characterized by mixing a solution containing carbonic acid and bicarbonate and iv) a solution containing lactoferrins (solution A) and v) a solution containing iron (solution B). A method for producing a carbonic acid and / or bicarbonate-iron-lactoferrin complex. However, at this time, the vi) iron ion molar concentration of the B solution is determined by vii) the molar concentration of carbonate ion and bicarbonate ion dissolved in a solution (reaction solution) in which the A solution and a part or all of the B solution are mixed. Less than 1/3 of the concentration
Solution iX) The molar concentration of lactoferrins is x) of solution B)
It is 1/15 to 1/1000 of the iron ion molar concentration. 1) Each molecule of lactoferrin contains 15 to 1000 molecules of iron and 15 or more molecules of carbonic acid and / or bicarbonate. 2) Prevents precipitation at room temperature at a pH of 2.1 to 9.0 for at least one month. ) No precipitation when heated. 4) No astringent taste unique to iron.